Project Summary/Abstract. We seek to identify novel therapeutic agents that are selectively toxic to cancer cells and that specifically sensitize tumors to radiation or chemotherapy. We have discovered that a cell-penetrating, lupus-derived autoantibody (3E10) increases the sensitivity of cancer cells to radiation and to DNA-targeted chemotherapy. Importantly, 3E10, by itself, is synthetically lethal to BRCA2- and PTEN-deficient cancer cells, but is otherwise non-toxic to cells in culture or to mice. The antibody also showed no detectable toxicity in humans when tested in a phase I clinical trial in lupus patients as a putative anti-idiotype vaccine. We previously determined 3E10 to be a potent inhibitor of homology-dependent repair (HDR), and we have now identified RAD51 as the functional target. We have also found that 3E10 is preferentially taken up in tumor tissue in vivo based on its mechanism of cell penetration, providing a further basis to pursue its development for cancer therapy. These new results provide the basis to enhance the potency of 3E10 (by directed mutation, affinity maturation, and multi-valent constructs) and to rationally develop therapeutic strategies by identifying synthetic lethal interactions (via unbiased shRNA dropout screen and interrogation of curated cancer cell lines) and determining synergies with other agents, as a prelude to pre-clinical animal tumor studies. We expect that 3E10 will be synthetic lethal to cancers deficient in DNA repair and damage response pathways. We also have developed a strategy to selectively target DNA repair inhibitors to tumors by exploiting the acidic tumor microenvironment. We will use a pH low insertion peptide (pHLIP) that inserts directionally across cell membranes at low pH and delivers cargoes selectively into tumor cells in vivo. Focusing on DNA-PK in the non-homologous end-joining pathway (NHEJ) of DNA repair, we will build on advances made in collaborative work to develop tumor-targeted antisense and small molecule inhibition of DNA-PK. We will incorporate next generation ?PNAs modified at the ? position to increase binding to RNA for potent antisense activity. This is based on our promising proof-of-concept studies published in Nature demonstrating the in vivo anti-tumor activity of pHLIP-PNA conjugates. We will also conjugate small molecule DNA-PK inhibitors to pHLIP, leveraging potent molecules that have not advanced to the clinic because of normal tissue toxicity, and conferring tumor selectivity. This work will provide a versatile platform to apply to other DNA repair targets. We have recently identified the oncometabolite, 2-hydroxyglutarate (2HG), as a new biomarker of deficient DNA repair in human malignancies. We found that elevated levels of 2HG confer a BRCAness phenotype of deficient HDR that renders cancer cells sensitive to synthetic lethal killing by PARP inhibitors and by 3E10. 2HG is produced by the neomorphic activity of isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations found in gliomas, leukemia, and other cancers. We will investigate the mechanism by which 2HG suppresses DNA repair and identify vulnerabilities that can be exploited for therapeutic gain in human tumors.